The broad objective of this application is to understand how host cells defend themselves against pore-forming toxins made by pathogenic bacteria. The long term objective is to use this information for development of novel therapeutics that protects humans against bacterial infection by boosting cellular defenses against pore-forming toxins. Pore-forming toxins are the largest single group of bacterial virulence factors, comprising >25% of all protein toxins made by bacteria. They are important for the in vivo virulence of many major bacterial pathogens of humans, including Staphylococcus aureus and Streptococci. Caenorhabditis elegans has emerged as an important genetically-tractable in vivo system for studying how animal cells defend against pore-forming toxins in a manner relevant to mammals. c-JUN N-terminal Kinase (JNK) mitogen-activated protein kinase (MAPK) is a central regulator of induced cellular defenses to pore-forming toxins in C. elegans, regulating half of all PFT-induced defenses. Specific Aim I will use forward and reverse genetics, genetic and biochemical tests, quantitative intoxication assays, and expression analyses to assemble as completely as possible the C. elegans JNK PFT-induced defense pathway, heading both upstream of JNK towards initial pore-formation and downstream of JNK into regulated defense subpathways. Specific Aim II involves the use of mammalian cells treated with a medically relevant pore-forming toxin to ascertain how well C. elegans defenses are conserved in mammalian cells. Specific Aim III uses a unique and powerful set of assays developed in C. elegans to study the actual mechanisms by which the JNK PFT-induced defense pathway protects cells against PFT attack. An additional goal of Specific Aim III is to uncover new cellular mechanisms for PFT defenses.